Recent advances in pharmacological, biochemical and molecular aspects of cannabinoids make it possible to explore the mechanism of actions in ways which were not available before. Characterization and cloning of cannabinoid receptors in brain and periphery led to the successful identification of the endogenous cannabinoid anandamide. Moreover, there is now evidence for a family of endogenous ligands rather than a single entity. The major endeavor of this project is to characterize the pharmacological actions of the endogenous cannabinoids and identify commonalties between delta9-THC and the endogenous cannabinoids. Newly discovered endogenous ligands will be assessed for their pharmacological profile using CB1 and CB2 receptor binding, a mouse behavioral model and drug discrimination. Studies will also be conducted in mice to determine the degree to which tolerance develops to the endogenous ligands. Cross- tolerance will allow us to verify which actions the endogenous ligands and delta9-THC share. An antagonist has not been available until the recent announcement by the research group at Sanofi that they had developed SR141716A, a cannabinoid antagonist. Since our preliminary studies show that this compound is effective in both antagonizing and reversing the effects of delta9-THC, we are confident that it will play a vital role in the characterization of endogenous ligands. Due to the fact that very little is known regarding this antagonist, a complete antagonistic profile will be determined. We will generate pA2 values for delta9-THC and the endogenous ligands to ascertain which actions are mediated through a common receptor. Based upon our previous experience, we know that tolerance development to synthetic cannabinoid analogs can result in receptor down-regulation and increased receptor mRNA levels. Thus, we will determine whether the endogenous ligands are capable of altering these systems in a similar fashion. Studies are proposed to determine whether the antagonist exhibits any pharmacological effects on its own, whether tolerance develops to these effects and whether receptor upregulation and decreased mRNA levels result from chronic administration. Finally, structure-activity relationship studies will be carried out for analogs of the endogenous ligands and the antagonist. As yet, we do not have a clear understanding of the receptor-ligand interactions which can accommodate agonists as structurally diverse as THC and arachidonic acid derivatives. Even more importantly, we do not know the structural properties of SR141716A which account for its antagonistic properties. The results from our studies will provide probes and valuable guidance to the other members of the Program Project.